Transcriptome analysis to compare alternative splicing between control and Ahed-deficient hematopoietic stem and progenitor cells (HSPCs) [RNA-Seq]

We discovered a novel functional gene "Ahed" by screening mutant embryonic stem cells (ESCs). We confirmed that Ahed deletion was disruptive to multiple hematopoietic lineages, and Ahed-deficient HSPCs were unable to reconstitute hematopoiesis in vivo. A search through the biophysical interactions of ORFeome-based complexes 3.0 (BioPlex 3.0) network database indicated a close correlation of Ahed protein in the RNA splicing process. Therefore, we conducted RNA-Seq analysis on triplicate differentiated populations comparing control and Ahed cKO FL LSK cells to verify the reproducibility of our findings. Skewed splicing patterns were observed in diverse transcripts for numerous genes, including PU.1, Ythdf2, Sel1l, and Zfp60, which are known to play important roles in hematopoiesis. We performed RNA-Seq analysis on triplicate differentiated populations comparing control or Ahed cKO Lin- Sca1+ c-Kit+ (LSK) cells in E14.5 fetal liver.本研究通过对突变型胚胎干细胞（Embryonic Stem Cells, ESCs）进行筛选，成功鉴定出一个全新的功能基因"Ahed"。实验证实，Ahed基因的敲除会破坏多个造血谱系，且Ahed缺陷型造血干细胞及祖细胞（Hematopoietic Stem and Progenitor Cells, HSPCs）无法在体内重建造血功能。通过检索基于开放读码框库的复合物3.0（BioPlex 3.0）网络数据库的生物物理相互作用数据，发现Ahed蛋白与RNA剪接过程存在紧密关联。为验证研究结果的可重复性，本研究对三份生物学重复的分化群体开展RNA测序（RNA-Seq）分析，以野生型对照组与Ahed条件性敲除（conditional knockout, cKO）胎肝LSK细胞为比对对象。研究观察到，包括PU.1、Ythdf2、Sel1l及Zfp60在内的诸多已知在造血过程中发挥关键作用的基因，其各类转录本均出现了剪接模式异常偏移的现象。本研究还针对E14.5胎肝中的Lin⁻Sca1⁺c-Kit⁺（LSK）细胞，对其三份生物学重复的分化群体开展RNA测序分析，以野生型对照组与Ahed条件性敲除细胞为比对对象。